Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential Nicole Bennardo1 2 Amanda Gunn1 2 Anita Cheng1 Paul Hasty3 Jeremy M Stark1 2 1 Department of Cancer Biology Division of Radiation Biology Beckman Research Institute of the City of Hope Duarte California United States of America 2 City of Hope Graduate School of Biological Sciences Duarte California United States of America 3 Department of Molecular Medicine Institute of Biotechnology The University of Texas Health Science Center at San Antonio San Antonio Texas United States of America Abstract To characterize the repair pathways of chromosome double strand breaks DSBs one approach involves monitoring the repair of site specific DSBs generated by rare cutting endonucleases such as I SceI Using this method we first describe the roles of Ercc1 Msh2 Nbs1 Xrcc4 and Brca1 in a set of distinct repair events Subsequently we considered that the outcome of such assays could be influenced by the persistent nature of I SceI induced DSBs in that end joining EJ products that restore the I SceI site are prone to repeated cutting To address this aspect of repair we modified I SceI induced DSBs by coexpressing I SceI with a non processive 39 exonuclease Trex2 which we predicted would cause partial degradation of I SceI 39 overhangs We find that Trex2 expression facilitates the formation of I SceI resistant EJ products which reduces the potential for repeated cutting by I SceI and hence limits the persistence of I SceI induced DSBs Using this approach we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations EJ between distal ends of two tandem DSBs single strand annealing and alternative NHEJ In contrast Trex2 expression does not inhibit homology directed repair These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss Furthermore we find that individual genetic factors play distinct roles during repair of non cohesive DSB ends that are generated via co expression of I SceI with Trex2 Citation Bennardo N Gunn A Cheng A Hasty P Stark JM 2009 Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential PLoS Genet 5 10 e1000683 doi 10 1371 journal pgen 1000683 Editor Gregory P Copenhaver The University of North Carolina at Chapel Hill United States of America Received June 25 2009 Accepted September 15 2009 Published October 16 2009 Copyright 2009 Bennardo et al This is an open access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited Funding This work was supported by NIH grant RO1CA120954 to JMS The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript Competing Interests The authors have declared that no competing interests exist E mail jstark coh org is single strand annealing SSA which also causes deletions with homology at repair junctions but involves extensive regions of homology 6 In addition for each of these pathways loss of correct end pairing during the repair of multiple simultaneous DSBs can lead to chromosomal rearrangements For instance EJ between distal ends of two tandem DSBs Distal EJ results in loss of the chromosomal segment between the DSBs To characterize the genetic factors that influence these pathways one approach involves analyzing repair of site specific DSBs in mammalian cells such as those generated by the rare cutting endonuclease I SceI For instance using this approach HDR SSA and Alt NHEJ were shown to be promoted by CtIP and Nbs1 7 10 which are factors implicated in the formation of ssDNA via end resection 9 11 As well the strand exchange factors Rad51 Brca2 were found to promote HDR and suppress SSA 12 13 and a number of additional genetic factors have been found to promote HDR 14 Other studies have addressed the influence of factors involved in NHEJ during V D J recombination including Ku and Xrcc4 Ligase IV For example Ku Xrcc4 deficient cells show higher HDR 15 and Ku deficient cells show elevated SSA and Alt NHEJ 5 In addition Ku and Xrcc4 have been shown to promote EJ that restores the I SceI site measured as EJ between distal ends of two tandem I SceI induced DSBs S DEJ 16 17 To further address the process of DSB repair pathway choice in mammalian cells we have developed this two part study In the Introduction Chromosome double strand breaks DSBs can be repaired by a number of mechanisms with a variety of mutagenic consequences 1 In the context of ionizing radiation IR therapy or chemotherapy that utilizes DSB inducing agents such DNA damage in non tumor cells could result in oncogenic mutations that cause secondary malignancies 2 Thus characterizing the factors and pathways that influence DSB repair will be important to develop therapeutic approaches that may limit the risk of secondary tumors and to understand the etiology of genome rearrangements associated with primary cancer development DSB repair pathways show a varying propensity for genetic loss A relatively precise form of repair is homology directed repair HDR that uses the identical sister chromatid as a template for Rad51 mediated strand invasion and nascent DNA synthesis 1 In contrast end joining EJ pathways are variably mutagenic depending on the extent of end processing and the fidelity of endpairing For instance EJ via the V D J recombination nonhomologous end joining NHEJ machinery has the potential to be precise especially when DSB ends can be ligated without significant processing 3 However Ku independent EJ Alternative NHEJ Alt NHEJ often leads to deletion mutations which are predominantly associated with short stretches of homology microhomology at repair junctions 4 5 Similar to Alt NHEJ PLoS Genetics www plosgenetics org 1 October 2009 Volume 5 Issue 10 e1000683 DSB Persistence and Repair somally integrated inactive GFP cassette where the structure of each reporter is designed such that repair of the DSB by a specific pathway results in restoration of the GFP cassette For instance three reporters were designed to measure distinct end joining EJ events as described previously 7 and summarized below First the EJ5 GFP reporter measures end joining between distal ends of two tandem I SceI
View Full Document
Unlocking...